U.S. patent application number 10/766783 was filed with the patent office on 2005-07-28 for lactoferrin-treated filament materials.
Invention is credited to Naidu, A. Satyanarayan.
Application Number | 20050163825 10/766783 |
Document ID | / |
Family ID | 34795741 |
Filed Date | 2005-07-28 |
United States Patent
Application |
20050163825 |
Kind Code |
A1 |
Naidu, A. Satyanarayan |
July 28, 2005 |
Lactoferrin-treated filament materials
Abstract
Disclosed is a filament composition having a surface for
reducing microbial contamination having a surface for reducing
microbial contamination comprising a filament material, such as a
dental floss or a suture material, and lactoferrin.
Inventors: |
Naidu, A. Satyanarayan;
(Diamond Bar, CA) |
Correspondence
Address: |
KENNETH J. HOVET
NORDMAN, CORMANY, HAIR & COMPTON
P.O. BOX 9100
1000 TOWN CENTER DRIVE
OXNARD
CA
93031-9100
US
|
Family ID: |
34795741 |
Appl. No.: |
10/766783 |
Filed: |
January 27, 2004 |
Current U.S.
Class: |
424/443 ;
514/17.2; 514/2.5; 514/44R; 514/5.7; 514/54; 514/56; 514/9.3 |
Current CPC
Class: |
A01N 63/50 20200101;
A61L 2300/404 20130101; A61L 17/005 20130101; D06M 16/00 20130101;
A61C 15/041 20130101; A61K 38/40 20130101; A61L 2300/606 20130101;
A61L 2300/252 20130101; D06M 16/003 20130101; A01N 63/50 20200101;
A01N 25/08 20130101; A01N 25/34 20130101 |
Class at
Publication: |
424/443 ;
514/006; 514/002; 514/044; 514/054; 514/056 |
International
Class: |
A61K 038/40; A61K
048/00 |
Claims
What I claim is:
1. A filament composition having a surface for reducing microbial
contamination comprising a filament material and lactoferrin.
2. The filament composition in accordance with claim 1 wherein at
least some of the lactoferrin is immobilized on a biologically
active substrate via the N-terminus region of the lactoferrin.
3. The filament composition in accordance with claim 2 wherein the
ratio of immobilized lactoferrin to free lactoferrin is from about
1:1 to about 1:500.
4. The filament composition in accordance with claim 2 wherein the
ratio of immobilized lactoferrin to free lactoferrin is from about
1:4 to about 1:100.
5. The filament composition in accordance with claim 2 wherein the
ratio of immobilized lactoferrin to free lactoferrin is about
1:20.
6. The filament composition in accordance with claim 2 wherein the
biologically active substrate is a protein, a polysaccharide, a
nucleic acid, a nucleotide or a lipid.
7. The filament composition in accordance with claim 2 wherein the
biologically active substrate is galactose-rich polysaccharide,
collagen, gelatin, fibronectin, casein, mucin, heparan-sulfate,
carrageenan, pectin, deoxyribonucleic acid, adenosine triphosphate
or a triglyceride.
8. The filament composition in accordance with claim 2 wherein the
concentration of the lactoferrin on the surface of the filament
composition for reducing microbial contamination is from about
0.0001 to about 10 mg/square inch.
9. The filament composition in accordance with claim 2 wherein the
concentration of the lactoferrin on the surface of the filament
composition for reducing microbial contamination is from about 0.01
to about 1 mg/sq. inch.
10. The filament composition in accordance with claim 2 wherein the
filament material is a monofilament or a multifilament
material.
11. The filament composition in accordance with claim 2 wherein the
surface of the filament composition for reducing microbial
contamination has a coating containing the lactoferrin.
12. The filament composition in accordance with claim 2 wherein the
filament material is covalently bonded to the lactoferrin.
13. A dental floss composition having a surface for reducing
microbial contamination comprising a dental floss material and
lactoferrin.
14. The dental floss composition in accordance with claim 13
wherein at least some of the lactoferrin is immobilized on a
biologically active substrate via the N-terminus region of the
lactoferrin.
15. The dental floss composition in accordance with claim 14
wherein the ratio of immobilized lactoferrin to free lactoferrin is
from about 1:4 to about 1:100.
16. The filament composition in accordance with claim 14 wherein
the biologically active substrate is a protein, a polysaccharide, a
nucleic acid, a nucleotide or a lipid.
17. The filament composition in accordance with claim 14 wherein
the biologically active substrate is galactose-rich polysaccharide,
collagen, gelatin, fibronectin, casein, mucin, heparan-sulfate,
carrageenan, pectin, deoxyribonucleic acid, adenosine triphosphate
or a triglyceride.
18. The dental floss composition in accordance with claim 14
wherein the concentration of the lactoferrin on the surface of the
dental floss composition for reducing microbial contamination is
from about 0.0001 to about 10 mg/square inch.
19. The dental floss composition in accordance with claim 14
wherein the concentration of the lactoferrin on the surface of the
dental floss composition for reducing microbial contamination is
from about 0.01 to about 1 mg/sq. inch.
20. The dental floss dental composition in accordance with claim 14
wherein the dental floss material is a monofilament material, a
multifilament material or a tape.
21. The dental floss composition in accordance with claim 14
wherein the dental floss material is a multifilament material.
22. The dental floss composition in accordance with claim 14
wherein the surface of the dental floss composition for reducing
microbial contamination has a coating containing the
lactoferrin.
23. The dental floss composition in accordance with claim 22
wherein the coating is a pH sensitive wax or polymeric coating.
24. The dental floss composition of claim 14 wherein the dental
floss material has a layer of a hydrophilic polymer treated with
the lactoferrin and a permeation enhancer.
25. The dental floss composition in accordance with claim 14
wherein the dental floss material is covalently bonded to the
lactoferrin.
26. A suture composition having a surface for reducing microbial
contamination comprising a suture material and lactoferrin.
27. The suture composition in accordance with claim 26 wherein at
least some of the lactoferrin is immobilized on a biologically
active substrate via the N-terminus region of the lactoferrin.
28. The suture composition in accordance with claim 27 wherein the
ratio of immobilized Lactoferrin to free Lactoferrin is from about
1:4 to about 1:100.
29. The suture composition in accordance with claim 27 wherein the
biologically active substrate is a protein, a polysaccharide, a
nucleic acid, a nucleotide or a lipid.
30. The suture composition in accordance with claim 27 wherein the
biologically active substrate is galactose-rich polysaccharide,
collagen, gelatin, fibronectin, casein, mucin, heparan-sulfate,
carrageenan, pectin, deoxyribonucleic acid, adenosine triphosphate
or a triglyceride.
31. The suture composition in accordance with claim 27 wherein the
concentration of the Lactoferrin on the surface of the suture
composition for reducing microbial contamination is from about
0.0001 to about 10 mg/square inch.
32. The suture composition in accordance with claim 27 wherein the
concentration of the lactoferrin on the surface of the suture
composition for reducing microbial contamination is from about 0.01
to about 1 mg/sq. inch.
33. The suture composition in accordance with claim 27 wherein the
suture material is a monofilament material or a multifilament
material.
34. The suture composition in accordance with claim 27 wherein the
suture material is a monofilament material.
35. The suture composition in accordance with claim 27 wherein the
suture material is a multifilament material.
36. The suture composition in accordance with claim 27 wherein the
surface of the suture composition for reducing microbial
contamination has a coating containing the lactoferrin.
37. The suture composition in accordance with claim 27 wherein the
dental floss material is covalently bonded to the lactoferrin.
Description
[0001] Throughout this application various publications are
referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference in this application
in order to more fully describe the state of the art to which this
invention pertains.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates to the chemical arts. In particular,
it relates to treated filament materials having antimicrobial
properties.
[0004] 2. Discussion of the Related Art
[0005] Once pathogenic microbes invade the natural barriers of the
body they present the risk of infection to the invaded host. Two
common means of access by bacteria are through the oral cavity,
potentially causing a variety of periodontal disease and at the
site of sutured tissue, as in the case of wounds and/or surgical
intervention sites, where the body's normal immune defenses are
breached.
[0006] The teeth and the mouth are vulnerable to many diseases,
infections, and disorders. It is well known that a good regime of
oral hygiene is the best preventative measure against cavities or
dental caries, gum or periodontal disease, viral and fungal
infections of the oral cavity, and other dental disorders.
[0007] Gum disease is an infection of the tissues surrounding and
supporting the teeth. It is a major cause of tooth loss. In the
early stages of gum disease, called gingivitis, the gums can become
red, swollen and bleed easily. At this stage the disease is still
reversible and can usually be eliminated by brushing and
flossing.
[0008] Periodontitis is the more advanced stage of gum disease in
which the gums and bones that support the teeth become severely
damaged. Periodontitis can be caused by unremoved plaque. Plaque is
a film of bacteria and mucous that grows on the tooth surface. Some
of the bacteria in the plaque make acids which cause tooth decay.
Other kinds of bacteria in the plaque make toxins which cause gum
disease. The plaque causes the gums to become irritated and
inflamed. The irritated gum tissue can separate from the teeth and
form spaces called pockets. Bacteria move into the pockets and
continue to cause irritation. Left untreated the process can
continue until the bone and other tooth-supporting tissues are
destroyed. Various agents are currently used to control plaque
formation and other microbial infections in the mouth, but,
unfortunately, they suffer from a variety of drawbacks.
[0009] Flossing is an extremely important component of proper
dental hygiene. Dental flosses have long been used effectively to
clean the spaces between the teeth and under the gum margin. One
example of a dental floss is disclosed in U.S. Pat. No. 3,800,812.
The art of present commercial dental flosses is well exemplified by
U.S. Pat. No. 4,414,990, U.S. Pat. No. 4,033,365 and U.S. Pat. No.
3,943,949 which disclose the use of various
non-polytetrafluoroethylene filaments as a floss; U.S. Pat. No.
5,033,488 which discloses a floss with a single strand of expanded
polytetrafluoroethylene that has been coated with a
microcrystalline wax; and U.S. Pat. No. 6,270,890 which discloses a
dental floss of both polytetrafluoroethylene filaments and
non-polytetrafluoroethylene filaments.
[0010] To increase the effectiveness of the floss, some flosses
have included certain medicinal ingredients, such as fluoride
compounds, to protect the tooth enamel from acid attack. For
example, U.S. Pat. No. 3,830,246, U.S. Pat. No. 3,897,795, U.S.
Pat. No. 4,215,478 and U.S. Pat. No. 3,771,536 disclose dental
flosses which include a fluoride compound to aid in the delivery of
the fluoride to the tooth surface between adjacent teeth.
Bactericides have also been used in connection with dental floss to
inhibit periodontal disease. For example, U.S. Pat. No. 6,159,447
and U.S. Pat. No. 6,482,396 disclose compositions for treating
bacterial colonization and diseases in an oral cavity. The
medicinal components have typically been applied as a coating to
the dental floss.
[0011] It is also known to have dental flosses that include other
kinds of ingredients. For example, U.S. Pat. No. 4,033,365
discloses a floss designed to retain flavorants over a long period
of time through the use of non-wax polymeric coatings containing
spray-dried flavor particles. U.S. Pat. No. 3,943,949 discloses a
dental floss-like material in the form of a bundle of natural or
synthetic fibers, such as nylon. The floss is coated with various
waxes, including microcrystalline wax, to reduce the friction of
the floss against the tooth surface. The wax coating is disclosed
as containing a spray-dried flavorant to be dispersed during
use.
[0012] Postoperative surgical site infections occur in
approximately 2.5% of all patients who undergo surgical procedures.
It is quite common that some type of suture is utilized. While the
body's immune response normally is successful in preventing
microbial infection at the wound site, in the presence of foreign
matter, such as the suture, the probability of infection increases
significantly.
[0013] The primary mode of infection associated with a suture is
attachment of microorganisms, e.g., bacteria, to the suture,
followed by their growth and proliferation on the suture.
Subsequent release and migration of the microbial contaminant from
the microbe's original attachment and growth to tissue immediate to
and surrounding the suture results in an infection associated with
the suture. Once the microbes attach and establish themselves on
the suture, it is practically impossible to treat the infection
without actually removing and replacing the suture or other wound
closure material or device.
[0014] While antimicrobial substances bacteriocins, i.e.,
substances that in and of themselves are toxic to microorganisms
capable of causing infection at surgical sites, may be added to
suture, they typically have limitations. Many of the antimicrobial
substances are toxic to the patient, while others cause allergic
reactions. In addition, certain microorganisms are resistant to
such antimicrobial substances due to the development of defense
mechanisms that actually destroy the antimicrobial molecule.
[0015] Various products for use externally or internally with
humans or animals can serve to introduce bacterial, viral, fungal
or other undesirable infections. Such products include suture,
medical devices, surgical gloves and implements, catheters,
implants and other medical implements. To prevent such
contamination, such devices can be treated with an antimicrobial
agent. Known methods of preparing infection-resistant medical
devices have been proposed in U.S. Pat. Nos. 3,566,874; 3,674,901;
3,695,921; 3,705,938; 3,987,797; 4,024,871; 4,318,947; 4,381,380;
4,539,234; 4,612,337; 3,699,956; 4,054,139; 4,592,920; 4,603,152;
4,667,143 and 5,019,096. U.S. Pat. No. 5,607,681 discloses
anti-microbial compositions that can be impregnated on sutures and
dental floss.
[0016] Antimicrobial agents with selective toxicity for a specific
spectrum or range of pathogenic microorganisms are well known in
the art. One class of antimicrobial agents is the antibiotics,
which are compounds, synthesized and excreted by various
microorganisms, that are selectively toxic to other microorganisms,
specifically bacteria. In addition, some antibiotics can be
artificially modified to produce antimicrobial agents that are more
effective and/or more able to overcome antibiotic resistance.
[0017] PCT/US00/14818 describes an antimicrobial assay using
buffered solutions containing both free lactoferrin and lactoferrin
immobilized on a variety of substrates to block attachment
(microbial blocking activity) of various oral pathogens to
subepithelial matrix proteins, oromucoid cell line, hydroxyapatite
(HA) and denatured bases acrylic resin (DBAR) surfaces. Lactoferrin
is an antimicrobial iron-binding glycoprotein present in milk and
various mammalian secretions (including saliva, tears, mucus, and
seminal fluids). Crystallographic studies of LF indicate a bilobate
structure (N-terminus and C-terminus lobes) with one iron-binding
site in each lobe. LF has ability to reversibly bind two Fe.sup.3+
ions per lobe in coordination with two CO.sub.3.sup.2- ions. LF can
release the bound iron in a fully reversible manner, either on
exposure to lowered pH (below 4.0) or on receptor binding. This
high affinity for iron is linked to many of its biological
functions, including antimicrobial effects. Various laboratory
studies have reported that the structural integrity of LF is
critical for its antimicrobial effects against bacteria, fungi,
protozoa, and viruses.
[0018] However, the activity of LF, like the activity of most
proteins, is highly dependent on the three-dimensional or tertiary
structure of the protein. If the protein does not have the proper
conformation its activity is diminished or lost. LF's instability
limits it usefulness. Consequently, before LF can be used for
commercial application, it would be expected to become denatured or
inactivated, and lose its antimicrobial properties.
SUMMARY OF THE INVENTION
[0019] Now, in accordance with the invention there has been found a
filament composition having a surface for reducing microbial
contamination comprising a filament material, such as a dental
floss or a suture material, and lactoferrin. In preferred
embodiments, at least some of the lactoferrin is immobilized on a
biologically active substrate via the N-terminus region of the
lactoferrin. Preferably, the ratio of immobilized LF to free LF is
from about 1:1 to about 1:500, more preferably from about 1:4 to
about 1:100, and most preferably about 1:20. The concentration of
the LF on the surface of the filament composition for reducing
microbial contamination is typically from about 0.0001 to about 10
mg/square inch and preferably from about 0.01 to about 1 mg/sq.
inch.
[0020] Representative filament materials include monofilament
materials, multifilament materials and tapes. Representative
biologically active substrates include proteins, polysaccharides,
nucleic acids, nucleotides or lipids. Examples of preferred
biologically active substrates include collagen, gelatin,
fibronectin, casein, mucin, heparan-sulfate, carrageenan, pectin,
deoxyribonucleic acid, adenosine triphosphate or triglycerides.
[0021] In some embodiments, the surface of the filament composition
has a coating containing the lactoferrin. In other embodiments, the
filament material is covalently bonded to the lactoferrin.
[0022] In some embodiments, the filament material is a dental floss
material having a pH sensitive wax or polymeric coating. In other
embodiments, the filament material is a dental floss material
having a layer of a hydrophilic polymer treated with the
lactoferrin and a permeation enhancer.
[0023] The following detailed description is provided to aid those
skilled in the art in practicing the present invention. Even so,
this detailed description should not be construed to unduly limit
the present invention as modifications and variations in the
embodiments discussed herein can be made by those of ordinary skill
in the art without departing from the spirit or scope of the
present inventive discovery.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0024] In accordance with the present invention there has been
discovered filament materials that have been treated with LF.
Representative filament materials include dental floss materials
and suture materials. In preferred embodiments, at least some of
the LF is immobilized on a substrate, via attachment of its
amino-terminus (N-terminus), leaving its carbon-terminus
(C-terminus) free.
[0025] Any suitable dental floss or suture material can be used in
accordance with the invention. Useful dental flosses come in
different forms and include monofilaments, multi-filaments, as well
as tapes. As used in the present invention the term filament
material includes such monofilaments, multi-filaments, and tape
materials and the terms "dental floss" and "dental floss material"
are meant to include all of the filamentous forms of floss, the
tape forms of dental floss and equivalents of the same.
[0026] As a tape, the dental floss typically has a denier of about
1200 to 3000 or more. As multi-filaments, the individual filaments
typically have a denier of about 100 to 800. The advantage of a
multi-filament over a tape or a monofilament is that, in use, the
filaments of a multi-filament floss splay and assist in the removal
of food particles, debris and plaque from between the teeth and
under the gum line. This enhanced cleaning comes from the splayed
filaments each rubbing the surface of a tooth. The use of a
plurality of filaments appears to exhibit an increased removal of
certain particles and plaque.
[0027] In preferred embodiments, the dental floss material, as it
is formed, will undergo a twisting to form the filaments into a
more cohesive form. There can be from about one to five twists per
inch of filament.
[0028] In some embodiments, the filament material has a coating
containing the LF. Suitable coating formulation(s) can
advantageously contain, in addition to LF, an appropriate carrier
(s). For example, the skilled practitioner can employ as a carrier
a non-toxic polymeric resin, additionally containing an effective
amount of LF, which resin can be used to coat the surface of the
treated filament material, hardening in place upon it.
[0029] In some embodiments, the suture material is coated with a
coating, preferably a pH sensitive coating, which will release the
LF under conditions of use. Representative coatings include wax and
polymeric coatings, such as polyethylene coatings, or acrylic
polymer coatings, including Eudragit-L or Eudragit-S coatings, or
cellulose coatings, including ethyl cellulose coatings. Ethyl
cellulose, for example, is amphoteric and dissolves to release the
LF at either acidic or basic pH. Acrylic polymer coatings have
various pH sensitivities. For example, Eudragit-S dissolves at
above pH 7.0 to release the LF. Alternatively, the filament
material can be sprayed, brushed or soaked with a wax coating
material containing the LF.
[0030] Alternatively, the dental floss material can comprise an
inner layer containing the LF, a permeation enhancer, such as a
bile salt or fusidate, and a hydrophilic polymer, such as
hydroxypropyl cellulose, hydroxypropyl methylcellulose,
hydroxyethylcellulose, dextran, pectin, polyvinyl pyrrolidone,
starch, gelatin, or any of a number of other polymers known to be
useful for this purpose. This inner layer can have one surface
adapted to contact and adhere to the moist mucosal tissue of the
oral cavity and may have an opposing surface adhering to an
overlying non-adhesive inert layer. Optionally, such dental floss
material may be produced in a manner such that the inner layer also
contains additional binding agents, flavoring agents or fillers.
Some useful systems employ a non-ionic detergent along with a
permeation enhancer. These examples are merely illustrative of
available delivery technology that may be used with the dental
floss material of the present invention and are not intended to
represent of be limitations of the present invention.
[0031] The LF dental floss material of the present invention is
optionally packaged on a spool, reel or disposed in a handle or
other applicator. It may then be dispensed in any of the
conventional manners.
[0032] Useful suture materials may be of any appropriate natural or
synthetic composition. These materials may be used as single
filament strands, i.e., monofilament sutures, or as multifilament
strands in a braided, twisted or other multifilament construction.
Examples of suitable materials include nylon, polypropylene, steel,
polyvinyl fluoride, linen, cotton, silk and polyesters. Natural
materials such as silk, cotton, linen, and the like, of course do
not lend themselves to the fabrication of monofilament sutures and
are accordingly generally used in one of the multifilament
constructions.
[0033] Conventionally, sutures have been manufactured of
non-degradable materials like nylon and polypropylene or degradable
or absorbable materials like polyglycolic acid and copolymers of
glycolic acid and lactic acid. Biodegradable or absorbable sutures
are designed to decompose in the tissue environment of the patient,
desirably after a time when wound recovery has occurred to an
extent that their strength is no longer necessary. This allows
patients to not have to return to a physician to have their suture
removed. Absorbable sutures are manufactured from natural or
synthetic materials. Some of the earliest absorbable sutures were
made of natural material, such as collagenous material taken from
sheep intestines. Such sutures are still in use today and are
commonly referred to as "catgut" or simply "gut" sutures or
ligatures. Gut sutures may be prepared in the form of threads or
strands.
[0034] Absorbable sutures made from synthetic material are
typically extruded in continuous lengths can be used in
monofilament form. Common synthetic monofilament sutures include
polyethylene terephthalate, polypropylene, polyethylene,
polyglycolides, polylactides and nylon. Different circumstances for
application of sutures demand different properties. Important
properties that have to be adjusted, depending on the type of
application of a suture, are knot strength, slip, tensile strength,
pliability and the like. Such monofilament sutures are often
preferred for many applications due to their inherent smoothness
and noncapillarity to body fluids.
[0035] Absorbable sutures typically can be made for short term or
long term use. The classification short term generally refers to
sutures which retain at least about 20 percent of their original
strength for three weeks, with the suture being essentially
absorbed in the body within about 60 to 90 days. Absorbable
multifilament sutures such as DEXON sutures (made from glycolide
homopolymer and commercially available from Davis & Geck,
Danbury, Conn.), VICRYL sutures (made from a copolymer of glycolide
and lactide and commercially available from Ethicon, Inc.,
Sommerville, N.J.), and POLYSORB sutures (also made from a
copolymer of glycolide and lactide and commercially available from
United States Surgical Corporation, Norwalk, Conn.) are known in
the industry as short term absorbable sutures. Long term absorbable
sutures are generally classified retaining at least about 20
percent of their original strength for six or more weeks, with the
suture being essentially absorbed in the body within about 180
days. For example, PDS II and MONOCRYL (commercially available from
Ethicon, Inc., Sommerville, N.J.), MAXON suture (commercially
available from Davis & Geck, Danbury, Conn.) and BIOSYN
(commercially available from United States Surgical Corporation)
are synthetic monofilament that reportedly generally fit a
long-term absorption profile.
[0036] The terms "LF", "LF protein", and "LF peptide" are used
interchangeably herein. The LF useful in accordance with the
materials and methods of the present invention include or contain
glycosylated or unglycosylated LF peptides. A full length LF
peptide sequence has about 600 to about 800 contiguous amino acids.
For example, native human LF is about 703 amino acids long; native
bovine LF is about 651 amino acids long. Other useful mammalian LF
sequences are of various but similar lengths. Useful LF peptides
include full length native LF peptides and also include LF peptides
lacking one to about eleven contiguous amino acids from the extreme
end of the N-terminus region or the extreme end of the C-terminus
region of a native LF peptide amino acid sequence. Also useful are
LF peptides having sequences variant in one or more amino acid
residues compared to a native LF sequence, but that remain at least
partially functional. The term "functional", when used herein as a
modifier of LF protein (s) or peptide (s), generally refers to a
polypeptide that retains the antimicrobial activity attributed to
native LF amino acid sequences. In the context of immobilized LF,
the term "functional", when used herein as a modifier of LF protein
(s) or peptide (s), generally refers to a polypeptide that exhibits
both the ability to bind at its N-terminus to a substrate, i.e.,
become immobilized, and also retains the antimicrobial activity
attributed to native LF amino acid sequences. Thus, the term LF
encompasses functional LF having a variant amino acid sequence.
[0037] The LF can be, but is not necessarily, of homologous origin
with respect to the vertebrate subject to which it is used or
administered, in accordance with the present methods. Thus, for
example, in accordance with the inventive method, LF of human
origin functions to reduce or inhibit microbial contamination or
growth in or on human and/or non-human vertebrates on sutured
tissue surfaces, tooth and/or gum or other periodontal surfaces.
Similarly, bovine LF can be used to treat either bovine or
non-bovine subjects. However, for use on human or non-human
vertebrates, in vivo, homologous LF is preferred to avoid adverse
immunoreactions.
[0038] The LF peptide can be isolated from mammalian sources
(humans, cows, sows, mares, transgenic animals, and the like),
biological secretions, such as colostrum, transitional milk,
matured milk, milk in later lactation, and the like, or processed
products thereof such as skim milk and whey. Also useful for the
isolation of LF is well-known recombinant DNA technology, whereby
cloned LF-encoding genes are expressed in procaryotic and/or
eucaryotic cells. The LF peptide is isolated by any conventional
method, such as by chromatography, ion-exchanger, molecular-sieve
or affinity column. Suitable LF also is commercially available from
DMV International Nutritionals, the Netherlands; Morinaga Milk
Company, Japan; BioPole, Belgium; and Glanbia, USA.
[0039] The LF can be immobilized on any suitable biologically
active substrate, i.e., any substrate that can bind the N-terminus
region of the LF without adversely affecting the LFs antimicrobial
activity. In preferred embodiments, LF is immobilized on a
naturally occurring substrate. Suitable substrates include
proteins, polysaccharides, nucleic acids, nucleotides, and lipids.
Preferred substrates include collagen, gelatin, fibronectin,
casein, mucin, heparan-sulfate, carrageenan, pectin,
deoxyribonucleic acid, adenosine triphosphate or a
triglyceride.
[0040] Another preferred substrate is a galactose-rich
polysaccharide (GRP). Galactose-rich polysaccharides are known in
the art as water-soluble extracts of agar that contain a majority
of galactose residues and/or galactose derivatives, which can be
substituted or non-substituted. (Gerlach, D. et al., Identification
of a novel lectin in Streptococcus pyogenes and its possible role
in bacterial adherence to pharyngeal cells, Current Microbiology
28: 331-38 [1994]). Suitable galactose-rich polysaccharides include
galactose derivatives comprising galactose, anhydrogalactose,
2-Ome-galactose, and 4-Ome-galactose, among others. Galactose-rich
polysaccharides can also contain a minority of other sugar and
non-sugar components, including residues of nitrogen-containing
non-sugar compounds and/or sulfated residues. The galactose-rich
polysaccharides can be purchased or extracted from commercial agars
by known methods. (E.g., Gerlach, D. et al. [1994]; Naidu, A. S.,
Agar, Chapter 16, In: Natural Food Antimicrobial Systems, A. S.
Naidu (ed.), CRC Press, Inc., pp. 417-27 (2000). Other suitable
biologically active substrates include proteins, such as collagen,
denatured collagen (gelatin), fibronectin, and casein;
polysaccharides, such as mucin, heparan-sulfates, carrageenan, and
cellulose; nucleic acids and their nucleotides, such as
deoxyribonucleic acid and adenosine triphosphate; and lipids such
as triglycerides.
[0041] The LF is immobilized on the substrate using any suitable
technique. For example, LF can be immobilized simply by mixing
isolated LF with the biologically active substrate in a suitable
medium, such as deionized water. The immobilization process is
dependent on the quality of the substrate as well as the quality of
the LF. For example, in most of the commercially available
lactoferrins, there is variation in the level of impurities (range:
4-20%), degree of non-specific cidal activity (range: 20-40%), and
extent of protein denaturation (range: 10-25%). Consequently, the
amount of substrate and the amount of LF to be used in the
immobilization reaction will depend, inter alia, on the choice of
starting materials. The immobilization technique and the amounts of
substrate and LF are readily determined by a skilled artisan
without undue experimentation.
[0042] In some embodiments, the immobilized LF (Im-LF) is combined
with free LF. Mixtures of Im-LF and free LF are formed by adding
excess LF to the substrate. Preferably, the ratio of the LF to free
LF is from about 1:1 to about 1:500, more preferably from about 1:4
to about 1:100, and most preferably about 1:20.
[0043] The LF can be incorporated into the filament material by any
suitable means, such as by including in a coating or a hydrophilic
polymer layer, or by otherwise treating the filament material. In
some embodiments, a covalent linkage between the filament material
and the LF is produced. For example, some treatments result in
conjugation of the LF to the polymeric material through, for
example, diazo bonds or amide bonds. The filament material is
treated at any time prior to the use. For example, the LF,
preferably Im-LF, can be combined with the filament material during
the manufacture of the suture or floss on the filament or
filaments, prior to the twisting, braiding or other manufacturing
treatment of the filaments.
[0044] LF may also be applied to the floss or suture material after
its typical manufacture. An LF-containing coating formulation can
be applied by any suitable method. Representative methods include
spraying, brushing or soaking the filament material with a
dispersion of the LF, including dispersions incorporated into a
microsphere or particle (coated or not).
[0045] Dispersions useful in accordance with the invention can be
prepared in various ways. A first way is by forming a solution
containing the LF, most preferably an aqueous solution containing
Im-LF, along with an emulsifier. Representative emulsifiers include
mono-, di-triglyceride compounds, glycerol, phosphatidyl
ethanolamine, phosphatidyl choline, or lecithin. One embodiment
includes a mixture of mono- and diglyceride compounds. Suitable,
commercially available mixtures (containing 35-45% monoglycerides)
include GRUENAU Mono & Diglycerides, and C. G. 340-E (Bavaria
Corporation, Altamonte Springs, Fla.). In embodiments in which LF
is immobilized on a triglyceride or other lipid substrate, the
Im-LF can be held in solution, if the solution has the properties
of bile (i.e., is a solution of mixed micelles with bile salt
added), or the solution contains a detergent or a solvent (e.g. the
solution contains Tween).
[0046] A second way of preparing dispersions useful in accordance
with the invention is by forming an emulsion containing the LF,
i.e., by forming a two-phase system in which a first liquid,
containing LF, is dispersed in the form of small globules
throughout a second liquid that is immiscible with the first
liquid. (Swinyard and Lowenthal, "Pharmaceutical Necessities"
Remington's Pharmaceutical Sciences, 17.sup.th ed., A R Gennaro
(Ed), Philadelphia College of Pharmacy and Science, 1985, p. 1296).
Aqueous emulsions containing a second, hydrophobic liquid phase are
preferred. The concentration of LF in emulsions is typically from
about 10.0 to about 0.001 wt. %, preferably from about 5.0 to about
0.05 wt. %, and more preferably from about 2.0 to about 0.2 wt.
%.
[0047] A third way of preparing dispersions useful in accordance
with the invention is by forming a suspension of a solid phase
containing the LF, either dispersed within a liquid phase, such as
a colloid suspension of LF, or dispersed among other solids (e.g.,
microcrystalline suspension), the composition thus having the form
of a powder or a granular solid. The concentration of LF in such
dispersions is typically from about 10.0 to about 0.001 wt. %,
preferably from about 5.0 to about 0.05 wt. %, and more preferably
from about 2.0 to about 0.2 wt. %.
[0048] In those embodiments where the dispersion is applied as a
liquid spray, the dispersion can also contain from about 10.0 to
about 0.001 wt. %, preferably from about 5.0 to about 0.05 wt. %,
and more preferably from about 2.0 to about 0.2 wt. % of a
film-forming agent. Suitable film-forming agents include
carrageenan, gelatin or collagen (Type-I and Type-II).
[0049] Alternatively, the LF can be applied to the filament
material, especially a polymeric dental floss material, by
enmeshing, implanting, or impregnating the LF within the polymeric
material, by means known to the artisan skilled in the art. In
still other embodiments, the LF may be applied to the floss or
suture material as they are dispensed for use. One skilled in the
art will recognize that there are additional methods for treating
the suture or dental floss material with the LF.
[0050] The surface of the inventive filament compositions contains
an amount of LF effective to reduce microbial contamination.
Preferably, the concentration of the LF on the surface for reducing
microbial contamination is from about 0.0001 to about 10 mg/square
inch., more preferably from about 0.01 to about 1 mg/sq. inch. This
is sufficient concentration to inhibit the growth and/or adhesion
of microbes on the surface contacted with the treated filament
material.
[0051] The LF composition used to treat the filament material can
include conventional additives. As is appropriate for the
filament's use as suture or dental floss, representative additives
may include one or more of the following, medicament(s), including
additional antimicrobial agent(s), flavorant(s), nutrient(s),
solvent(s), vehicle(s), adjuvant(s), excipient(s), binder(s),
thickener(s), suspending agent(s), or filler substance(s). Useful
additives include, but are not limited to, solid, semisolid or
liquid glucose, lactose, sucrose, or polymeric substances like
starch or dextran.
[0052] The additives can be applied to the filaments by any
suitable method. Representative methods include applying the
additive to the filament as a liquid and then drying the additive
onto the filaments. Alternately, the additives can be applied to
the filaments as a solid with the aid of a binder. Suitable binders
include polyvinyl alcohol, and in particular, polyvinyl alcohol in
combination with polyethylene glycol.
[0053] Useful additional antimicrobial agents include acid
antimicrobials, such as lactic acid, acetic acid, citric acid,
sorbic acids; ionic antimicrobials, such as polyphosphate,
nitrites; sulfur compounds; chlorocides; ozone; or a natural,
synthetic, or an semi-synthetic antibiotic agent, such as neomycin,
metronidazole, teicoplanin, vancomycin, ciprofloxacin, doxycycline,
tetracycline, augmentin, erythromycin, chloramphenicol, cephalexin
(e.g., Keflex), penicillin, ampicillin, kanamycin, rifamycin,
rifaximin, rifampin, clindamycin, trimethoprim, a 4-amino
salicylate compound, a 5-aminosalicylate compound, a sulfonamide
compound, a betalactam compound, an aminoglycoside compound, a
macrolide compound, or a quinolone compound.
[0054] In dental floss compositions, a preferred form of flavorant
is a spray dried flavorant. The flavorant can be essentially any
flavor but is preferably a peppermint and/or spearmint. This can be
applied to the filaments using a non-wax polymeric binder as is
described in U.S. Pat. No. 4,033,365. If the dental floss
composition is wax coated, the spray dried flavorant can be applied
to the still molten wax.
[0055] The inventive compositions are useful against a wide variety
of bacteria, such as, but not limited to pathogenic and
non-pathogenic strains of:
[0056] (A) Gram-negative facultative anaerobes of the enteric
group, for example, Escherichia coli; Helicobacter pylori;
Salmonella spp., including Salmonella typhimurium, Salmonella
typhi, Salmonella enteritidis, Salmonella abony, Salmonella dublin,
Salmonella hartford, Salmonella kentucky, Salmonella panama,
Salmonella pullorum, Salmonella rostock, Salmonella thompson,
Salmonella virschow; Enterobacter spp., such as Enterobacter
aerogenes; Klebsiella pneumonie; Shigella spp., such as Shigella
dysenteriae or Shigella flexneri; Vibrio spp., including Vibrio
cholerae; Yersinia enterocolitica and Yersinia pestis.
[0057] (B) Gram-negative aerobic motile rods, such as Bordetella
pertussis; Campylobacter jejuni; and Pseudomonas spp., such as
Pseudomonas aeruginosa;
[0058] (C) Gram-negative aerobic non-motile rods, such as Brucella
spp.; Legionella pneumophila; and Francisella tularensis;
[0059] (D) Gram-positive bacteria, including coccoid forms such as
Staphylococcus spp., such as Staphylococcus aureus, Staphylococcus
epidermidis; Streptococcus spp., such as Streptococcus pyogenes,
Streptococcus pneumoniae, Streptococcus mutans, Streptococccus
sanguis; Pediococcus acne; and bacillary forms such as Bacillus
spp., including Bacillus anthracis, Bacillus cereus, Bacillus
pumilus, Bacillus subtilis; Clostridium spp., including Clostridium
difficile, Clostridium tetani, Clostridium botulinum, Clostridium
perfringens; and Listeria monocytogenes;
[0060] (E) Periodontal pathogens, such as Actinobacillus
actinomycetemcomitans, Porphyromonas gingivalis; Prevotella spp.,
such as Prevotella intermedia.
[0061] Further, the inventive compositions are useful against
fungal pathogens including dermatophytes, such as Epidermophyton
spp.; Microsporium spp.; and Trichophyton spp.; systemic
mycopathogens, such as Blastomyces spp.; Coccidiodes spp.;
Cryptococcus neoformans; Histoplasma spp.; and yeasts, such as
Candida albicans. Still further, the inventive compositions are
useful against protozoan parasites, such as Entamoeba histolytica;
Naegleria flowleri; Giardia lamblia; Leishmania spp.; Trichomonas
vaginalis; Trypanosoma spp.; Plasmodium spp.; and Taxoplasma
spp.
[0062] And still further, the inventive compositions and methods
are also useful against viral pathogens, including herpes viruses,
such as HHV-6 and HHV-8, Cytomegalovirus (CMV); Epstein-Barr virus
(EBV); Herpes Simplex viruses (HSV); Varicella viruses; Picorna
viruses such as Coxsackie viruses; Hepatitis A virus; Rhinoviruses;
Retroviruses, such as the Rotaviruses, Influenza, and Parainfluenza
viruses.
[0063] The inventive compositions are especially useful in treating
or preventing infections, including clostridial infections, at any
oral or periodontal site or tissue of a vertebrate. Such
clostridial infections include, but are not limited to, gangrene or
tetanus, caused, respectively, by Clostridium perfringens and
Clostridium tetani, when these species grow in wounds and damaged
tissues with low oxygen tension.
[0064] Moreover, the inventive compositions can act synergistically
to potentiate some antibiotic agents, including beta-lactams,
chloramphenicol, aminoglycosides, clindamycin, vancomycin,
sulfonamides, trimethoprim, rifampin, tetracyclines, metronidazole,
quinolones, erythromycin, and other macrolides.
[0065] The present invention includes a method of preventing or
inhibiting the growth and/or adhesion of a microbe in or on a
vertebrate subject, including a human subject. The human subject
can be an infant, child, or adult. The method is also useful for
veterinary purposes. The present method is useful for treating any
non-human vertebrate including, but not limited to a wild, exotic,
domestic, or farm animal. For example, the method is useful for
treating an appropriate species of reptile, amphibian, avian, fish,
shark or a mammal such as a non-human primate, mouse, rat, rabbit,
gerbil, hamster, canine, feline, ovine, bovine, porcine, pachyderm,
equine, or marine mammal.
[0066] The inventive dental floss compositions and related
materials are of particular use on oral surfaces, such as oral
tissues, as well as in oral fluids, including blood, lymph, saliva,
gastric juice, and mucus, and interspaces within oral surfaces. The
dental floss material may be used in the conventional manner well
known in the art. Typically a section or segment of dental floss is
used in an action commonly referred to as "flossing," wherein the
dental floss is passed between the contact points of two teeth and
the floss is pulled or moved along the tooth surface proximate to
thee gum tissue as is appropriate for the specific subject. The
subject's stage of oral hygiene and/or periodontal disease will
dictate the specific manner of flossing or use for the dental floss
material of the present invention.
[0067] As further example, in some application such as veterinary
uses or for difficult treatment in human subjects, a practitioner
may prefer to wrap a segment of the dental floss material around a
tooth or fix the segment temporarily in the area of the tooth, gum
or other periodontal surface for some indications or situations,
rather than using a typical flossing action. The inactive dental
floss composition may be used with implements such as dental floss
holders or applicators, as are known in the art or other aids in
the use and application of the dental floss composition of the
invention.
[0068] The suture compositions are useful in reducing microbial
infection, adhesion, and/or contamination on biological surfaces,
including, cell surfaces, membranes, mucosa, epithelia, lumenal
surfaces, of a human or non-human vertebrate, including oral
epithelium, or any other surface at an oral body site or site
having sutured tissue. The inventive suture material may be used in
the conventional manners well known in the art. A typical example
includes attaching suture to a surgical needle by methods also well
known in the art and passing the needle and suture through the
tissue of a wound or tissue opening to create a closure or
attachment or occlusion as intended by the practitioner.
[0069] The foregoing applications for the methods and compositions
of the present invention are illustrative and by no means
exhaustive. The invention will now be described in greater detail
by reference to the following non-limiting examples. All weights
are based on percent weight/volume unless otherwise clearly
indicated.
EXAMPLES
[0070] An antimicrobial assay was performed to demonstrate the
inhibitory effect of LF on a variety of bacterial strains.
[0071] Preparation of LF:
[0072] A 2% (wt./vol) Im-LF/LF mixture was prepared by dissolving
2.0 g LF isolated from cow milk (DMV International Nutritionals,
Veghel, The Netherlands) in a 100-ml sterile buffer solution formed
of deionized water containing 1 mM EDTA (Versene NAJ from Dow
Chemicals, Freeport, Tex.); 10 mM NaHCO.sub.3 (Fisher, Fairlawn,
N.J.); and 1 mM NaCl (Sigma Chemicals, St. Louis, Mo.). After
adjusting the pH to 8.2 (with NaHCO.sub.3), food-grade pectin (0.02
g; CU 201 from Herbstreith & Fox, Neurenburg, Germany) was
added to the solution at room temperature with gentle stirring for
ninety minutes. There resulted the partial immobilization of the
dissolved LF. The formation of Im-LF was confirmed by gel
filtration chromatography using Sephacryl S-200 HR column.
[0073] Preparation of LF-coated Suture Filaments
[0074] Two different suture materials, a polypropylene-type
(Prolene Blue Monofilament, Ethicon Inc.) and a silk-type
(Black-braided with control release, Ethicon, Inc.) were coated
with the Im-LF/LF mixture as described above. Sterile suture
filaments of ca.1 cm length were immersed in the Im-LF/LF mixture
for 1-h, dried in sterile petri plates. The concentration of the
Im-LF/LF mixture soaked into the filament material was measured
using a quantitative enzyme-linked immunosorbant assay (ELISA)
commercial kit for LF (Bethyl Laboratories, Montgomery, Tex.). The
levels of LF in each 1-cm of soaked filament was in the range of
ca. 10-200 .mu.g.
[0075] Bacterial Test Strains:
[0076] Four different bacterial strains common to skin, skin
infections, and post-operative wound infections, i.e.,
Staphylococcus aureus ATCC12660, Staphylococcus epidermidis
ATCC12228, Pseudomonas aeruginosa ATCC27583, and Escherichia coli
ATCC43895, were used for the testing. All four test strains were
grown in tryptic soy broth (TSB) at 37.degree. C.
[0077] Bacterial Growth-Inhibition Properties of LF-Treated Suture
Filaments:
[0078] Bacterial growth-inhibition properties of LF coated suture
filaments were measured using a microbial growth impedance
detection assay (GIDA). A Bactometer.RTM. Microbial Monitoring
System Model-128 (bioMerieux Vitek, Hazelwood, Mo.) was used to
monitor bacterial growth by measuring impedance signals in the
cultivation media.
[0079] GIDA's were performed in sixteen wells. 1-ml TSB was added
to each well. Suture filaments treated with the IM-LF/LF mixture as
described above were immersed in 10-mL suspensions of the bacterial
test bacterial strains (containing .about.10.sup.4 bacteria/mL) for
10-min. Each of the filaments was gently removed from a suspension
and placed into one of the wells containing TSB. Addition of
untreated suture filaments, either with exposure or without
exposure to one of the bacterial test strains to wells containing
TSB served as controls for growth and sterility, respectively. The
inoculated wells (final volume: 1-mL) were incubated at 37.degree.
C., and impedance changes in the media were monitored by the
Bactometer.RTM. at 6-min intervals for 48-h. Bacterial growth
curves were graphically displayed as percent changes of impedance
signals versus incubation time. The amount of time required to
cause a significant deviation from baseline impedance value was
defined as the "detection time" (DT). The difference in DT values
between growth control and test samples was considered as the
"stasis" (growth-inhibition) time.
[0080] As seen in TABLE-1, all four bacterial strains demonstrated
interaction (carry-through) with suture filaments. Accordingly, the
suture-bound bacteria proliferated in TSB and gave an impedance
signal in the GIDA. The impedance DT values ranged from 4.0 to 6.9
hours for the polypropylene sutures and LF treatment of these
sutures extended the DT values in the range of 11.0 to 23.5 hours.
This indicated that LF-treated sutures elicited an effective
bacteriostasis ranging from +7.0 to +18.1-h. Similarly, LF
treatment of silk sutures also caused bacteriostasis ranging from
+7.3 to +13.9-h for the four bacterial test strains.
1 TABLE 1 Impedance detection time in hours (Stasis in +hours)
Polypropylene suture Silk suture Bacterial challenge Untreated LF
treated Untreated LF treated S. aureus ATCC12660 5.6 11.7 (+6.1-h)
6.4 13.7 (+7.3-h) S. epidermidis ATCC12228 6.9 15.9 (+9.0-h) 7.2
17.1 (+9.9-h) P. aeruginosa ATCC27853 5.4 23.5 (+18.1-h) 5.1 19.0
(+13.9-h) E. coli ATCC43895 4.0 11.0 (+7.0-h) 4.7 14.3 (+9.6-h)
Each data point represents an average value for quadruplicate
readings and the standard deviation for each average value is less
than 0.1
[0081] Bacterial Adhesion-Inhibition Properties of LF-Coated Suture
Filaments:
[0082] Bacterial adhesion/inhibition properties of LF-coated suture
filaments was measured using a .sup.3H-thymidine-labeled bacterial
adhesion assay. For radiolabeling of bacteria, a 50-L inoculum of
overnight culture of Staphylococcus aureus ATCC12660,
Staphylococcus epidermidis ATCC12228, Pseudomonas aeruginosa
ATCC27583, and Escherichia coli ATCC43895 grown in TSB was
re-inoculated in a sterile 10-mL of TSB tube containing
.sup.3H-thymidine (20 .mu.ci). All the four test strains were grown
at 37.degree. C. to exponential phase (about 5-7 h) to allow
optimum uptake and incorporation of .sup.3H-thymidine into their
bacterial DNA. .sup.3H-thymidine labeled bacterial cells were
harvested by centrifugation at 7,500.times.g, washed and
resuspended in phosphate buffered saline (PBS, pH 7.2). A
correlation curve was generated for each test strain between the
degree of thymidine labeling (scintillation counts measured as
disintegration per minute; DPM), bacterial viability (measured as
total viable plate counts) and total cell counts (OD measurement at
600 nm). The density of bacterial suspension was optically adjusted
to 1.0 OD at 600 nm (corresponding to .about.10.sup.9 cells/mL) and
further diluted in PBS to a final density of 10.sup.6 cells/mL for
adhesion experiments.
[0083] LF treated suture filaments were immersed in a 10-mL
suspension of .sup.3H-thymidine-labeled test bacterial strains
(containing 10.sup.6 bacteria/mL) for 1-min. Each of the filaments
was gently removed and placed in a scintillation vial containing
2-mL homogenizer (Scintiges.TM., Fisher) and incubated overnight in
a 50.degree. C. water bath. After total digestion of the suture
filament, 10-mL of scintillation cocktail (ScintiSafe.TM. Gel,
Fisher) was dispensed into the vial and thoroughly mixed. After
settling and clarification of the mixture, radioactivity was
measured using a liquid scintillation analyzer (Tri-Carb 2100
TR.RTM., Packard Inc.). Addition of untreated suture filaments
either exposure or without exposure to .sup.3H-thymidine-labele- d
test bacteria to scintillation vials served as controls for
bacterial attachment and background radioactivity,
respectively.
[0084] As seen in TABLE-2, treatment of sutures with LF resulted in
adhesion-inhibition of all four test strains in the range of
96.2-99.4% for the polypropylene sutures and 96.3-97.6% for the
silk sutures compared to their untreated counterparts.
2 TABLE 2 Radioactivity in DPM (% adhesion-inhibition)
Polypropylene suture Silk suture Bacterial challenge Untreated LF
treated Untreated LF treated S. aureus ATCC12660 4,387 167 (96.2%)
3,465 119 (96.6%) S. epidermidis ATCC12228 3,768 94 (97.5%) 2,998
106 (96.5%) P. aeruginosa ATCC27853 7,921 51 (99.4%) 8,763 328
(96.3%) E. coli ATCC43895 6,543 72 (98.9%) 5,939 145 (97.6%) Each
data point represents an average value for quadruplicate
readings
* * * * *